The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1␣ (HIF-1␣)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription.The basic helix-loop-helix PAS (Per-Arnt-Sim) (bHLH-PAS) protein family is a class of transcription factors with members present in vertebrates and invertebrates. These proteins contain a bHLH domain, a motif found in many transcription factors which undergo dimerization for function (for a review, see reference 42). The bHLH motif is present near the amino terminus of the protein and is contiguous with a second dimerization domain known as the PAS domain. The PAS domain, designated a conserved region among PER, ARNT (aryl hydrocarbon receptor nuclear translocator), and SIM, shows amino acid similarity over a stretch of 250 to 300 amino acids and contains degenerate hydrophobic repeats of approximately 50 amino acids each. Members of the bHLH-PAS protein family include (i) both subunits of the dioxin receptor complex, AHR (aryl hydrocarbon receptor) and ARNT, involved in the inducible expression of the CYP1A1 gene by xenobiotics (5, 14, 26); (ii) a close homolog of ARNT, called ARNT-2, which has similar protein association and DNA binding properties to ARNT (12,16,25); (iii) hypoxiainducible factor 1␣ (Hif-1␣), a trans-acting factor that, as a dimer with ARNT, mediates the genetic response to low oxygen t...
Protein tyrosine phosphatase-1B (PTP-1B) is a negative regulator of insulin signaling. It is thought to carry out this role by interacting with and dephosphorylating the activated insulin receptor (IR). However, little is known regarding the nature of the cellular interaction between these proteins, especially because the IR is localized to the plasma membrane and PTP-1B to the endoplasmic reticulum. Using confocal microscopy and fluorescence resonance energy transfer (FRET), the interaction between PTP-1B and the IR was examined in co-transfected human embryonic kidney 293 cells. Biological activities were not significantly affected for either PTP-1B or the IR with the fusion of W1B-green fluorescent protein (GFP) to the N terminus of PTP-1B (W1B-PTP-1B) or the fusion of Topaz-GFP to the C terminus of the IR (Topaz-IR). FRET between W1B and Topaz was monitored in cells transfected with either wild type PTP-1B (W1B-PTP-1B) or the substrate-trapping form PTP-1B D181A (W1B-PTP-1B D181A ) and Topaz-IR. Co-expression of W1B-PTP-1B with Topaz-IR resulted in distribution of Topaz-IR to the plasma membrane, but no FRET was obtained upon insulin treatment. In contrast, co-expression of W1B-PTP-1B D181A with Topaz-IR caused an increase in cytosolic Topaz-IR fluorescence and, in some cells, a significant basal FRET signal, suggesting that PTP-1B is interacting with the IR during its synthesis. Stimulation of these cells with insulin resulted in a rapid induction of FRET that increased over time and was localized to a perinuclear spot. Co-expression of Topaz-IR with a GFP-labeled RhoB endosomal marker and treatment of the cells with insulin identified a perinuclear endosome compartment as the site of localization. Furthermore, the insulin-induced FRET could be prevented by the treatment of the cells with a specific PTP-1B inhibitor. These results suggest that PTP-1B appears not only to interact with and dephosphorylate the insulin-stimulated IR in a perinuclear endosome compartment but is also involved in maintaining the IR in a dephosphorylated state during its biosynthesis.The insulin receptor is a heterotetramer plasma membrane protein that consists of two extracellular ␣ subunits and two membrane-spanning intracellular  subunits that contain inherent tyrosine kinase domains (1). The receptor is initially synthesized as a single polypeptide ␣- proreceptor that undergoes processing in the endoplasmic reticulum (ER).1 While it is in the ER, the proreceptor dimerizes and undergoes disulfide bond formation, glycosylation, and proteolysis before transfer to the plasma membrane (2-4). Binding of insulin to the receptor on the plasma membrane results in activation of the -subunit tyrosine kinase activity, and, once activated, autophosphorylation on three critical tyrosine residues in the kinase activation loop occurs, resulting in full activation of the tyrosine kinase activity (1). The activated IR then begins to phosphorylate its various substrates to initiate the insulin signaling cascade but, at the same time, undergoes...
Chromosome 11p15.5 harbors a gene or genes involved in Beckwith-Wiedemann syndrome that confer(s) susceptibility to Wilms' tumor, rhabdomyosarcoma, and hepatoblastoma. We have previously identified a transcript at 1 lp15.5 which encodes a putative membrane transport protein, designated organic cation transporter-like 2 (ORCTL2), that shares homology with tetracycline resistance proteins and bacterial multidrug resistance proteins. In this report, we have investigated the transport properties of ORCTL2 and show that this protein can confer resistance to ehloroquine and quinidine when overexpressed in bacteria. Immunohistochemistry analyses performed with anti-ORCTL2 polycional antibodies on human renal sections indicate that ORCTL2 is localized on the apical membrane surface of the proximal tubules. These results suggest that ORCTL2 may play a role in the transport of chloroquine and quinidine related compounds in the kidney.
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